Group I intron self-splicing with adenosine: evidence for a single nucleoside-binding site

For self-splicing of Tetrahymena ribosomal RNA precursor, guanosine binding is required for 5' splice-site cleavage and exon ligation. Whether these two reactions use the same or different guanosine-binding sites has been debated. A double mutation in a previously identified guanosine-binding site within the intron resulted in preference for adenosine (or adenosine triphosphate) as the substrate for cleavage at the 5' splice site. However, splicing was blocked in the exon ligation step. Blockage was reversed by a change from guanine to adenine at the 3' splice site. These results indicate that a single determinant specifies nucleoside binding for both steps of splicing. Furthermore, it suggests that RNA could form an active site specific for adenosine triphosphate.     

Efficient trans-splicing of a yeast mitochondrial RNA group II intron implicates a strong 5' exon-intron interaction

The reaction mechanism for self-splicing introns requires the existence of a 5' exon binding site on the intron. Experimental evidence is now presented consistent with the existence of such a binding site by demonstrating efficient and accurate trans-self-splicing of a yeast mitochondrial group II intron. Partial and complete trans-splicing reactions take place in the absence of branch formation, part of the usual pathway of nuclear splicing and group II self-splicing. In addition to indicating the existence of a 5' exon binding site on the intron, the results have mechanistic implications for group II self-splicing and perhaps for nuclear splicing as well.     

Visualizing the higher order folding of a catalytic RNA molecule

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.     

Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility

The bacteriophage T4 nrdB gene, encoding nucleoside diphosphate reductase subunit B, contains a self-splicing group I intervening sequence. The nrdB intron was shown to be absent from the genomes of the closely related T-even phages T2 and T6. Evidence for variable intron distribution was provided by autocatalytic 32P-guanosine 5'-triphosphate labeling of T-even RNAs, DNA and RNA hybridization analyses, and DNA sequencing studies. The results indicate the nonessential nature of the intron in nrdB expression and phage viability. Furthermore, they suggest that either precise intron loss from T2 and T6 or lateral intron acquisition by T4 occurred since the evolution of these phages from a common ancestor. Intron movement in the course of T-even phage divergence raises provocative questions about the origin of these self-splicing elements in prokaryotes.     

An RNA processing activity that debranches RNA lariats

The excised introns of pre-messenger RNA's (pre-mRNA's) and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is covalently joined by a 2',5'-phosphodiester bond to a specific adenosine residue near the 3' end of the intron. A 2',5'-phosphodiesterase activity in HeLa cell extracts has been detected that debranches RNA lariats, converting them to linear RNA molecules by specific cleavage of the 2',5'-phosphodiester bond. This lariat debranching activity is distinct from previously reported 2',5'-phosphodiesterases with regard to its biochemical and substrate requirements as well as its stringent cleavage specificity. The debranching activity is observed only if the RNA lariats generated during in vitro processing are deproteinized and added back to the extract. These results suggest that during the normal in vitro splicing reaction the 2',5'-phosphodiester bond of RNA lariats is protected from cleavage by the lariat debranching activity.     

Ribonuclease P catalysis differs from ribosomal RNA self-splicing

Two RNA-catalyzed reactions have been described, the Tetrahymena self-splicing ribosomal RNA and ribonuclease P. The Tetrahymena self-splicing reaction proceeds through a transesterification cascade that is dependent upon nucleophilic attacks by ribose 3'-OH groups. Periodate oxidation of the catalytic (or substrate) RNA, which destroys the nucleophilicity of RNA 3' termini, did not inhibit ribonuclease P activity. Thus, catalysis by ribonuclease P differs from the self-splicing reaction.     

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.     

Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria

A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.     

Crystal structure of a naturally occurring dinucleoside monophosphate: uridylyl (3',5') adenosine hemihydrate

The crystal structure of uridylyl (3',5') adenosine hemihydrate has been analyzed by x-ray diffraction. The two independent molecules found in the asymmetric unit exhibit conformations that differ significantly from those found in double-helical RNA. The conformational information obtained from this analysis provides considerable insight into the possible conformations of nonhelical "loop" regions of transfer RNA, as well as single-stranded regions of nucleic acids in general.     

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.     

Fluorescent modification of adenosine 3',5'-monophosphate: spectroscopic properties and activity in enzyme systems

The synthesis of a highly fluorescent analog of adenosine 3',5'-monophosphate, namely, 1,N(6)-ethenoadenosine 3',5'-monophosphate, has provided a powerful probe for systems involving adenosine 3',5'-monophosphate. The potential utility of this analog is indicated by its long fluorescent lifetime, detectability at low concentration, and relatively long wavelength of excitation (300 nanometers). In protein kinase systems it is a highly acceptable substitute for adenosine 3',5'-monophosphate.     

A specific amino acid binding site composed of RNA

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.     

HSPC117 is the essential subunit of a human tRNA splicing ligase complex

Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.     

Neuroblastoma: drug-induced differentiation increases proportion of cytoplasmic RNA that contains polyadenylic acid

The production of cytoplasmic RNA that contains polyadenylic acid is increased, relative to total cytoplasmic RNA, in a neuroblastoma clone, NBE-(A), after induction of differentiation by 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, an inhibitor of adenosine 3',5'-monophosphate phosphodiesterase. The amount of RNA that contains polyadenylic acid in cytoplasm may be greater in such differentiated neuroblastoma cells than in proliferating control cells.